This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ABSTRACT: Treponema spp. are invasive. They are able to penetrate cell monolayers (Lux et al., 2002;Thomas et al., 1988) and other dense matrices due in part to their unique motility. Their motility is a consequence of the helical or wave-shaped cell body and the periplasmic flagellar filament location (Limberger, 1984;Ruby &Charon, 1998). Treponema denticola is the model for Treponema pallidum subsp. pallidum (the agent of syphilis), as well as cultivable and non-cultivable oral spirochetes associated with periodontitis. Genetic manipulation of T. denticola is feasible, due to the recent development of a gene targeted interruption technique. The gene-specific interruption in T. denticola has already enhanced our knowledge of the biology and the pathogenesis of treponemal organisms (Izard et al., 2001;Limberger et al., 1999). Shuttle vectors, used for complementation and expression of proteins, have also been recently developed (Chi et al., 1999;Chi et al., 2002;), Limberger et al., (unpublished data), as well as new selection methods (Limberger et al., unpublished data). The T. denticola genomic sequence is also available facilitating the proposed studies. Treponema denticola is the model for Treponema pallidum subsp. pallidum (the agent of syphilis), as well as cultivable and non-cultivable oral spirochetes associated with periodontitis. Syphilis is an acute and chronic sexually transmitted disease. Syphilitic patients also show an increased risk for the acquisition and transmission of HIV (Quinn et al., 1988;Stamm et al., 1988). Elimination of syphilis in the U.S.A. would require a better knowledge of the responsible agent, either directly or through model organisms, since it cannot be cultivated (St. Louis &Wasserheit, 1998). Periodontal diseases are experienced by millions of people in the United States. It is now recognized that chronic oral infections, such as adult periodontitis, may have long-term sequelae (Beck et al., 1996;Grau et al., 2004). A quantitative relationship between the number of Treponema cells and the severity of periodontal disease has been demonstrated (Armitage et al., 1982;Moter et al., 1998). The data on organization of the periplasm in Treponema bacteria incomplete. Rotating flagellar bundles are organized adjacent to the periplasm. Their spacing has so far been characterized only after the use of fixatives or by freeze-fracture. Electron tomography has recently brought to light the organization of the cytoplasmic filaments associated with cell division in Treponema (Izard et al., 2004). Future work will be done with a more-native preparation technique. Preliminary images of whole mounts of Treponema denticola plunge-frozen in culture medium (see below) are promising. We can expect much better definition of the internal features in tomograms of such specimens. The structural studies proposed will provide a more detailed analysis of the consequences on the cell biology and cytoskeleon of Treponema if flagellar components were to be used as drug target. The overall goal of our research is to understand the molecular mechanisms involved in Treponema motility. Motility allows them to penetrate dense media and cell layers, and thus is a critical aspect of their pathogenesis. The first set of studies aims to understand the organization of the periplasm in its native state, without the use of artifact-inducing fixatives, by means of electron tomography carried out on plunge-frozen, frozen-hydrated whole mounts. The second set of studies aims to identify the effect of the absence of flagella on periplasmic organization. The third set of studies will provide a dynamic view of flagellar formation and insertion during cell septation. Aim #1. To refine the model of mechanical and dynamic organization of the periplasmic flagella, measurements of cell structures will be obtained from 3D reconstructions of cell segments Understanding the periplasmic and flagellar architecture will provide an opportunity to test hypotheses related to the mechanistic events associated with cell motility. Multiple flagella rotate at high speed within the periplasm, and their spatial organization in action has not yet been deciphered. Rapid freezing will provide "snapshots" of the motion. Aim #2. To complement the model, the ultrastructural effect of flagella loss will be observed Structural and biochemical changes are present in mutant strains that lack flagella. Tomographic reconstructions will help us understand the relation between the flagellar apparatus and other cell features, including membrane integrity and peptidoglycan positioning. Aim #3. To study the 3D spatial positioning of flagellar basal bodies at the septum of the cell division site in various stages of division The goal is to identify a 3D pattern of flagellar insertion. Patterning is suggested by 2D data obtained after removal of the outer membrane. Tomography of frozen-hydrated whole-mounts should reveal patterns in the native state. To complement these analyses, further work on the flagella filament will include knockout mutagenesis of genes related to flagellar proteins. These include proteins in the core and outer layer of the filaments, as well as the protein associated with core protein modification by the short length glycosylation pathway. Further work would concern identification of the network of proteins associated with flagellar rotation and anchoring. Mutant and wild-type Treponema cells will be brought to the RVBC in culture medium and quick-frozen by plunging in liquid ethane. Tilt series will be collected at liquid nitrogen temperature, with zero-loss energy filtering, using the 400kV JEOL 4000 TEM. Many of the tilt series will be collected around two orthogonal axes, which results in a more isotropic reconstruction. Alignment (using gold markers as shown in the image above) and reconstruction, followed by visualization and 3-D measurement, will be done using software developed at the RVBC. Isolated flagella will be studied in a similar manner. This research may lead to questions about cellular sub-structure that cannot be answered at the level of resolution obtainable with whole-mounts. In this case, pellets of cells will be high-pressure frozen and electron tomography will be carried out using frozen-hydrated sections. Since these sections can be cut at 50 nm and thinner, the highest possible resolution can be obtained. References 1. The Institute for Genomic Research (TIGR) WWW.tigr.org. 2. rmitage, G. C., Dickinson, W. R., Jenderseck, R. S., Levine, S. M. &Chambers, D. W. (1982). Relationship between the percentage of subgingival spirochetes and the severity of periodontal disease. J Periodontol 53, 550-556. 3. Beck, J., Garcia, R., Heiss, G., Vokonas, P. S. &Offenbacher, S. (1996). Periodontal disease and cardiovascular disease. J Periodontol 67, 1123-1137. 4. Chi, B., Chauhan, S. &Kuramitsu, H. (1999). Development of a system for expressing heterologous genes in the oral spirochete Treponema denticola and its use in expression of the Treponema pallidum flaA gene. Infect Immun 67, 3653-3656. 5. Chi, B., Limberger, R. J. &Kuramitsu, H. K. (2002). Complementation of a Treponema denticola flgE mutant with a novel coumermycin A1-resistant T. denticola shuttle vector system. Infect Immun 70, 2233-2237. 6. Grau, A. J., Becher, H., Ziegler, C. M. &other authors (2004). Periodontal disease as a risk factor for ischemic stroke. Stroke 35, 496-501. 7. Izard, J., Samsonoff, W. A. &Limberger, R. J. (2001). Cytoplasmic filament-deficient mutant of Treponema denticola has pleiotropic defects. J Bacteriol 183, 1078-1084. 8. Izard, J., McEwen, B. F., Barnard, R. M., Portuese, T., Samsonoff, W. A. &Limberger, R. J. (2004). Tomographic reconstruction of treponemal cytoplasmic filaments reveals novel bridging and anchoring components. Mol Microbiol 51, 609-618. 9. Limberger, R. J. (1984).Periplasmic flagella of Treponema phagedenis. West Virginia University, Morgantown. 10. Limberger, R. J., Slivienski, L. L., Izard, J. &Samsonoff, W. A. (1999). Insertional inactivation of Treponema denticola tap1 results in a nonmotile mutant with elongated flagellar hooks. J Bacteriol 181, 3743-3750. 11. Lux, R., Sim, J. H., Tsai, J. P. &Shi, W. (2002). Construction and characterization of a cheA mutant of Treponema denticola. J Bacteriol 184, 3130-3134. 12. Moter, A., Hoenig, C., Choi, B. K., Riep, B. &G[unreadable]bel, U. B. (1998). Molecular epidemiology of oral treponemes associated with periodontal disease. J Clin Microbiol 36, 1399-1403. 13. Quinn, T. C., Glasser, D., Cannon, R. O. &other authors (1988). Human immunodeficiency virus infection among patients attending clinics for sexually transmitted diseases. N Engl J Med 318, 197-203. 14. Ruby, J. D. &Charon, N. W. (1998). Effect of temperature and viscosity on the motility of the spirochete Treponema denticola. FEMS Microbiol Lett 169, 251-254. 15. St. Louis, M. E. &Wasserheit, J. N. (1998). Elimination of syphilis in the United States. Science 281, 353-354. 16. Stamm, W. E., Handsfield, H. H., Rompalo, A. M., Ashley, R. L., Roberts, P. L. &Corey, L. (1988). The association between genital ulcer disease and acquisition of HIV infection in homosexual men. JAMA 260, 1429-1433. 17. Thomas, D. D., Navab, M., Haake, D. A., Fogelman, A. M., Miller, J. N. &Lovett, M. A. (1988). Treponema pallidum invades intracellular junctions of endothelial cell monolayers. Proc Natl Acad Sci U S A 8, 3608-3612. In the previous reporting period, to complement the wild-type data collected, we made four tomographic reconstructions of mutant spirochetes that lack flagellar filaments. In these, the periplasmic filaments, located in the cytoplasm just beneath the flagellar filaments in the periplasmic space, could clearly be seen. Looking again at the tomograms from the wild type, the periplasmic filaments could be seen there as well. Several interesting features, not previously described, were seen in reconstructions of dividing wild-type spirochetes. Because contrast in the reconstructions was good, and since it is difficult to interactively trace spiral features in serial slices, attempts were made to segment filaments based on image parameters. Surface-rendered models of interphase and dividing cells were made. Tomographic reconstructions of six wild-type and four flagella-less mutant spirochetes were made from plunge-frozen whole-mounts. For the first time, all the main features of T. denticola were seen in native, intact cells: periplasmic space, flagella, cytoplasmic filaments, and basal bodies. Of particular interest was the structure of the periplasmic space at the flagella, and how the peptidoglycan layer is accommodated there. Interesting new features were seen in dividing cells, and at the tips of the cells. In the previous reporting period, the following book chapter was published: + Izard, J., Limberger, R.J. (2006) Structural and genomic aspects contributing to Treponema architecture. Book Chapter in: Molecular and Cellular Biology of Treponemes and Pathogenesis of Treponemal Infection. S.A. Lukehart and J. Radolf (ed.) Horizon Scientific Press (2006).